Project Summary Vascularized composite tissue allotransplantation (VCTA), a clinical reality since 1998, still faces many challenges yet to be addressed with basic and translational research. As the number of patients who have undergone VCTA grows, so does our understanding of the unique immunologic demands of composite grafts and their propensity toward rejection. Acute rejection is extremely common in clinical VCTA, with a reported incidence of 85% in hand and 54.5% in face transplants, requiring steroids and increases in immunosuppression to salvage the grafts (1). The experience with chronic rejection is growing, but it remains a poorly described entity, and still less is known regarding potential interventions (4). An emerging interest in cellular therapies to prevent and fight allograft rejection has led to the exploration of T-regulatory cells (T-regs) as a method to modulate the immune response toward a more tolerogenic state (7). T-regs have been demonstrated to be instrumental in long-term graft survival, and they attenuate the long-term sequelae seen in cases of chronic rejection, including tissue fibrosis and intimal hyperplasia (8,11-15). Additionally, an exciting new cellular technology, chimeric antigen receptor (CAR) T-regs, could allow for efficient, targeted therapy for VCTA rejection. By administering these cells to VCTA recipients, rejection could be prevented or possibly reversed. This study employs a murine hindlimb transplant model with two specific aims. First, transplanted, immunosuppressed mice will be serially biopsied to study the kinetics as well as the cellular aspects of VCTA rejection. Biopsies will examine both the architecture of the tissues as well as the makeup of graft cellular infiltrates. Mice will also be observed clinically for signs of acute and chronic rejection. Second, transplanted mice with rejection will be treated with engineered CAR T-regs as salvage therapy. We hypothesize that CAR T-reg infusion will halt or even reverse tissue and cellular changes seen in allograft rejection. This project seeks to elucidate what is known about the dynamics and cellular processes of rejection in VCTA, especially with respect to the role of T-regs, as well as apply a targeted cellular intervention for salvage of rejected grafts. CAR T-regs have shown clinical promise in the field of oncology, and if they are found to be successful in an animal model, they could feasibly be translated to the treatment of rejection in human hand or face transplants (17). Such an advance can provide clinicians with a powerful tool for treating composite allografts threatened by rejection, as well as make strides toward achieving the ultimate goal of tolerance without the life-long use of immunosuppressive drugs.